Ndiff 227 medium

For nonspecific differentiation, ESCs were washed in -LIF medium and then seeded at the cell densities specified below in each assay, as well as passaged on day one after seeding. Cells were assayed at 24, 48, 60, and/or 72 hr according to the experiment. For neural differentiation, ESC were first maintained in NDiff 227 medium (N2B27, Clontech) supplemented with 3 μM CHIR99021 (Selleck Chemicals) and 1 μM PD184352 (Selleck Chemicals), defined as 2i, to promote self-renewal in the absence of serum and LIF. Differentiation occurred in N2B27 in the absence of 2i and assayed at 24, 48, and 72 hr. For definitive endoderm differentiation, ESCs were grown in DMEM supplemented with 1% FBS, PSG, and 5 μM IDE1 (Cayman Chemical Company) and assayed at 48 hr. For EpiLC differentiation, ESCs were grown in N2B27 supplemented with 20 ng/mL activin A (R and D Systems), 12 ng/mL bFGF (Thermo Fisher), and 1% KOSR (Thermo Fisher) as previously shown (Hayashi et al., 2011 (link)), and assayed at 72 hr. For verteporfin-related experiments, verteporfin was diluted in DMSO to a concentration of 1 mM and then further diluted in fresh media during media changes, and cells were protected from light with aluminum foil. HEK293T cells (ATCC CRL-3216) were cultured in DMEM supplemented with 10% FBS and PSG. All cells were grown at 37°C in the presence of 5% CO₂.

Male mouse embryonal (E7) carcinoma P19 cells⁶⁰ (link) (ATCC, CRL-1825) were maintained in Dulbecco’s modified Eagle medium with 4,500 mg/L of glucose supplemented with 100 units/mL of penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (FBS, Gemini Bio-Products). For culture, cells were incubated at 37°C and 5% CO₂ atmosphere. All experiments have been conducted with original ATCC cells not exceeding eight passages. To induce neuronal differentiation, 3 x 10⁵ cells were seeded in poly-L-lysine-coated 6-well plates in TaKaRa NDiff® 227 Medium (Takara) with retinoic acid (500 nM; Sigma) as previously described.⁶¹ (link) Media was changed daily up to 6 days after induction. Morphological changes were visualized using the brightfield channel of the Nikon Eclipse TS2-fl microscope (Nikon Instruments Inc.).